PROJECT SUMMARY Vibrio cholerae is the etiological agent of the disease cholera. Cholera is an acute diarrheal disease prevalent in developing countries and is a major global health burden. The V. cholerae lifecycle is defined by repeated transitions between the marine environment and the human host. With respect to the disease, ingested planktonic V. cholerae cells migrate through the mucosal layer to colonize the epithelium of the small intestine. Infections are self-limiting: At the end of the infection cycle, V. cholerae disperses back into the environment as a result of the severe diarrhea characteristic of the disease cholera. This lifecycle requires that V. cholerae repeatedly make the key decision to switch between a free-living planktonic state or to join with others to form multicellular communities. This decision is controlled by quorum sensing (QS), the process of bacterial cell-cell communication that relies on the production, release, and group-wide detection of extracellular signal molecules called autoinducers. QS allows bacteria to collectively alter their behavior based on local population density. Multicellular community formation in bacteria, including V. cholerae, is commonly studied in the context of biofilms: surface-bound communities held together by an extracellular matrix. I have discovered an alternative multicellular community formation program in V. cholerae. This rapid, aggregative community formation program occurs in the absence of cell-division (<30 min to completion), does not require components essential for V. cholerae surface-biofilm formation, and occurs in liquid instead of on a surface. Like the surface-biofilm program this aggregative community formation program requires QS, although the pattern of regulation is different. I have determined the mechanism underlying how QS controls this new program and I conducted a genetic screen to identify genes required for aggregative community formation. My screen revealed genes involved in flagellar synthesis, encoding transcription factors for nutrient deprivation/stress, and a gene unique to the current pandemic strain of V. cholerae. Many of these genes are required for V. cholerae virulence in a murine model of cholera. My results suggest that this new multicellular community formation program facilitates transmission from the human host lumen back to the marine environment, promotes long-term environmental persistence by privatizing public goods production by the collective, and provides selectivity in community formation. My long- term goal is to develop a mechanistic and biophysical understanding of this new program of multicellularity in V. cholerae. I will use the tools of bacterial genetics, microscopy, proteomics, and biochemistry to (1) Determine how extracellular proteases regulate the timing of aggregative community formation; (2) Identify the structural components required for aggregative community formation; (3) Explore whether the aggregative community formation program protects participating cells from the toxic effects of bile, a stressor that V. cholerae must face during human infection. This work will deliver insight into the V. cholerae lifecycle and may lead to new strategies for combatting this important human pathogen.